There has been rapid growth in recent years in apparatus and methodology for biochemical enterprise, particularly for cleavage, separation and analysis of macromolecules such as DNA, including human DNA. There are a number of uses, such as determining the presence of genetically induced conditions such as Sickle-Cell Anemia, Huntington's Chorea and others Fluorescent gel scanning has also become important as an identification tool. Genetic code from a human hair or a flake of skin can be matched to a single human being, supplementing older procedures, such as fingerprint analysis in criminal law.
Typically, macromolecules are separated and identified by electrophoresis. It is well known in the art that electrically charged macromolecules of different size and structure move at different velocities in a specific medium under the influence of an electrical field. Typical mediums are agarose slabs and polyacrylamide gel films.
Agarose is a material that can be dissolved in hot water, and the solution formed solidifies to a gel state on cooling, so slabs of agarose can be prepared by pouring warm, liquid material into a structural container and cooling it. Agarose is typically poured in a horizontal slab, open-face. Wells are molded into the upper surface of a slab for introduction of sample, and the slab is immersed in buffer during the electrophoresis operation, Because the slabs are poured in relatively thick sections, such as 5 mm., sample wells of sufficient volume may be formed,
Agarose gels are capable of resolving DNA fragments above about 150 base-pairs in size. Much smaller fragments and relatively small macromolecules of other kinds tend to move through agarose more or less unimpeded, so they cannot be resolved with agarose gel systems. For relatively small DNA fragments, below about 500 base-pairs, and for relatively small macromolecules of other kinds, polyacrylamide gels are useful. There is a broad area of overlap in the sizes of macromolecules for which agarose or polyacrylamide is useful.
To prepare a polyacrylamide gel, a solution is prepared with acrylamide, a cross-linking agent, and a buffer, and the catalyst is added just prior to pouring. Polyacrylamide is typically cast in very thin films (0.5 mm is typical), partly because this material requires more electrophoretic driving force than agarose, and i s therefore more subject to heating by the passage of current. Because temperature gradients effect macromolecule migration and distort separation patterns, the gels must also be very thin to promote uniform temperature. Moreover, the cross-linked material tends to swell on exposure to buffer solution, so gels are cast between glass plates for structural support and dimensional integrity, to provide minimal exposure to air during polymerization and to buffer solution after polymerization.
Acrylamide polymerization proceeds only in the absence of oxygen, so areas exposed to air do not polymerize to form the gel. Typically buffer contacts only the ends of the very thin polyacrylamide gel film in operation.
Because polyacrylamide gels are usually prepared in thin films, formation of vertical wells in a horizontal film, the well having the depth of the thickness of the film, as is done in agarose systems, is not a suitable arrangement. Such wells are much too small in volume for adequate sample for electrophoresis. For this and other reasons polyacrylamide gels have traditionally been used in vertical systems. Sample wells are cast in the end of the film by inserting a comb before pouring the gel film.
Because agarose gels are typically horizontal systems, scanning apparatus has been developed also in a horizontal format for analyzing separations while electrophoresis is performed. Similarly, since polyacrylamide gels are typically vertical systems, scanning apparatus has been developed for these systems in a vertical format for analyzing separations while electrophoresis is performed. So, to cover all of the range of macromolecule sizes that are typically of interest, it is usually necessary to have gel apparatus of two different kinds, and also scanning apparatus of two different kinds.
Some efforts have been made to provide polyacrylamide electrophoresis apparatus that can be operated in a horizontal mode with scanning apparatus usually used for agarose gels. One way that has been tried involves casting a polyacrylamide gel horizontally between two glass plates, and forming vertical wells at right angles to the gel film at one end. A problem with this arrangement is that the voltage providing the electrophoretic driving force has to be applied from the end of the wells to the end of the film at the opposite end from the sample wells, and the electrical field (and the sample) has to pass around a right angle at the bottom of the well to enter the film. This has been found in practice to be troublesome, and to cause distortions in the separation bands.
To minimize the range and scale of apparatus necessary to cover a suitably broad range of sizes of macromolecules with adequate resolution, what is needed is an apparatus for polyacrylamide gel electrophoresis that can be used with horizontal scanning apparatus, and which does not suffer from the problems of right angle sample injection and the non-uniform patterns that result.